Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis

Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.     

Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Ethanol induces apoptosis in cerebellar granule neurons by inhibiting insulin-like growth factor 1 signaling

The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. IGF-1 protected granule neurons from apoptosis in medium containing 5 mM K+. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K+, and failed to potentiate cell death in the presence of 5 mM K+. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15-20%) was observed at 1 mM and was half-maximal at 45 mM. The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

Gene-based neurotransmitter modulation in cerebellar granule neurons

The human glutamic acid decarboxylase (GAD) gene was transferred into rat cerebellar granule neurons. Following adenoviral-mediated gene transfer, nearly 100% of the neurons had transgene expression that persisted for the duration of their survival in culture. GABA levels were elevated both in the growth media and in lysates of GAD-modified granule neurons. In GAD-modified neurons, extracellular GABA levels steadily increased with time, whereas intracellular GABA levels peaked 10 days after gene transfer. GAD-modified neurons released both glutamate and GABA into the surrounding media before and after potassium-induced stimulation, but only the release of glutamate was sensitive to potassium stimulation. These data suggest that glutamatergic neurons, which initially contained no detectable GABA, can be genetically modified to release GABA constitutively.     

Ornithine decarboxylase activity during development of cerebellar granule neurons

Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K+ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K+ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K+ but chronically exposed to 10 mM lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity.     

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis

In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage.     

Characterization of an ecto-phosphorylated protein of cultured cerebellar granule neurons

Previous work identified the phosphorylation by extracellular ATP of an endogenous 45-kDa protein substrate and established the presence of ecto-protein kinase activity associated with cultured cerebellar granule neurons (Volonté et al.: J Neurochem 63:2028-2037, 1994). In this work, we characterize such ecto-phosphorylated 45-kDa protein substrate and its association with the cellular membrane. The total radioactive content of the 45-kDa protein is stable for the first 15 min after phosphorylation, and decreases about 70% in 30 min and 90% in approximately 2 hr. Rinsing the cells after the phosphorylating reaction causes a 50% removal of the incorporated radioactivity. Glycosidic residues are present on the 45-kDa ecto-protein, which is held in position on the cellular membrane through a specific glycosyl-phosphatidylinositol anchor. The extracellular incorporation of phosphate on the 45-kDa protein is not modulated by agents interfering with cytoskeleton stability, such as colchicine and taxol, or by gangliosides. The extracellular phosphorylation occurs mostly on serine residues, since the phosphate ester linkage is unstable at high pH and only antibodies raised against phosphoserine are capable of recognizing the 45-kDa ecto-protein.     

Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis

Some clues suggest that neuronal damage induces a secondary change of amyloid beta protein (Abeta) metabolism. We investigated this possibility by analyzing the secretion of Abeta and processing of its precursor protein (amyloid precursor protein, APP) in an in vitro model of neuronal apoptosis. Primary cultures of rat cerebellar granule neurons were metabolically labeled with [35S]methionine. Apoptosis was induced by shifting extracellular KCl concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarization-stimulated secretion. Constitutive and stimulated secretion media and cell lysates were immunoprecipitated with antibodies recognizing regions of Abeta, full-length APP, alpha- and beta-APP secreted forms. Immunoprecipitated proteins were separated by SDS/PAGE and quantitated with a PhosphorImager densitometer. Although intracellular full-length APP was not significantly changed after apoptosis, the monomeric and oligomeric forms of 4-kDa Abeta were 3-fold higher in depolarization-stimulated secretion compared with control neurons. Such increments were paralleled by a corresponding increase of the beta-APPs/alpha-APPs ratio in apoptotic secretion. Immunofluorescence studies performed with an antibody recognizing an epitope located in the Abeta sequence showed that the Abeta signal observed in the cytoplasm and in the Golgi apparatus of control neurons is uniformly redistributed in the condensed cytoplasm of apoptotic cells. These studies indicate that neuronal apoptosis is associated with a significant increase of metabolic products derived from beta-secretase cleavage and suggest that an overproduction of Abeta may be the consequence of neuronal damage from various causes.     

Serum deprivation-induced apoptosis in cultured hippocampi is prevented by kainate

Serum deprivation of hippocampal organotypic cultures induced cell death within 6 h in dentate gyrus granule cells and hilar interneurons whereas neurons from other hippocampal regions were spared. Dying neurons exhibited condensed chromatin in the nuclei, as revealed by cresyl violet, Hoescht staining, and electron microscopy. Cell death was abolished by cycloheximide. KA, an agonist of AMPA/KA receptors that induces depolarization, also prevented neuronal death. This effect was antagonized by the AMPA/KA receptor antagonist DNQX, but not by APV, an antagonist of NMDA receptors. PTX, a GABA(A) receptor antagonist, reduced neuronal death by 50% after serum withdrawal. These data indicate that protein synthesis-dependent programmed cell death (PCD) occurs in the dentate gyrus upon trophic support withdrawal and suggest that neuronal activity contributes to cellular homeostasis.     

Cytotoxic action of lindane in cerebellar granule neurons is mediated by interaction with inducible GABA(B) receptors

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (gamma-HCH, lindane) was studied in cultured mouse cerebellar granule neurons maintained in the presence or absence of the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). The cells were exposed for 24 hr to lindane (30-300 microM) in the culture medium. Changes in mitochondrial function were investigated by using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The results showed that lindane-induced cytotoxicity was concentration-dependent. In cerebellar granule cells not treated with THIP, lindane-induced cytotoxicity did not appear to be related to GABA(A) or GABA(B) receptors. However, in THIP-treated cultures, lindane-induced cytotoxicity was found to be mediated by an action of the insecticide on GABA receptors. In the latter case, GABA reduced the lindane-induced cytotoxicity, but the protective effect was not potentiated by flunitrazepam. The GABA(A) receptor agonist muscimol (50 microM) also protected the THIP-treated cultures against lindane-induced cytotoxicity. In addition, the GABA(B) receptor agonist R(+)baclofen protected the cells from lindane-induced cytotoxicity and the effect of baclofen was blocked by GABA(B) receptor antagonists. Pertussis toxin was found to reverse the protective effect of baclofen only at the highest lindane concentration (300 microM). The lindane-induced cytotoxicity could be partly explained as being secondary to excitotoxicity as a mixture of the excitatory amino acid receptor antagonists APV (D-(-)-2-amino-5-phosphonopentanoate) and CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) shifted the concentration-response curve for lindane-induced cytotoxicity to the right. It is suggested that the cytotoxic effects of lindane in THIP-treated cerebellar granule neurons are primarily related to an action of lindane on GABA(B) receptors and to a lesser extent on inducible low-affinity, benzodiazepine insensitive GABA(A) receptors.     

The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+

Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.     

Elevated intracellular calcium levels in cerebellar granule neurons of weaver mice

Weaver mice carry a mutation in the pore domain of the Girk2 (Kcnj6) gene. The mutation causes GIRK2 containing channels to lose ion selectivity and to become constitutively active. It is not known how this alteration in ion channel activity causes in cerebellar granule cells the defects in neurite extension, cell migration and induction of cell death that are characteristic of weaver mice. One possibility is that the mutation causes an inability to regulate intracellular calcium levels properly. We tested this hypothesis by measuring intracellular calcium levels in granule cells and Purkinje cells in slices from the cerebellum of weaver mice. We report here that weaver mice have increases in resting calcium levels in their granule cells, which may account for the multiple effects of the weaver mutation upon these cells.     

Attenuation of cell death mediated by membrane depolarization different from that by exogenous BDNF in cultured mouse cerebellar granule cells

Membrane depolarization accompanying calcium (Ca2+) influx into neurons is thought to play an essential role in controlling the survival and death of cultured mouse cerebellar granule cells (CGCs). In this study, we sequentially controlled the survival and death of CGCs in culture and monitored the expression of several kinds of genes including brain-derived neurotrophic factor (BDNF) gene. Deprivation and subsequent induction of membrane depolarization by lowering and re-elevating the extracellular concentration of potassium chloride, respectively, led to death of CGCs and then to an attenuation of the death process depending upon the Ca2+ influx into CGCs through voltage-dependent calcium channels (VDCCs). De novo protein synthesis was critical for attenuating the death of non-depolarized CGCs. Accompanying this attenuation was an activation of c-fos and BDNF genes and an inactivation of c-jun and neurotrophin-3 (NT-3) genes. The attenuation of cell death mediated by exogenous BDNF was only partial compared to that by membrane depolarization, suggesting that not only BDNF but also other factors could be involved in the membrane depolarization-mediated attenuation of death of CGCs. In good agreement with this observation, the mode of activation of c-fos, c-jun, BDNF and NT-3 genes induced by exogenous BDNF was different from that induced by membrane depolarization. Thus, membrane depolarization effectively attenuates the death of non-depolarized CGCs, the mode of which seems to be different from that mediated by BDNF alone.     

A possible role for PKC delta in cerebellar granule cells apoptosis

Cerebellar granule cells (CGC) undergo massive DNA fragmentation, an apoptotic marker, in 8-day-old rat cerebellum. In vitro, they survive in the presence of depolarizing concentrations of KCl. Bisindolylmaleimide, a specific PKC inhibitor, blocks CGC apoptosis in vitro. Here I show that PKC delta, which has been involved in apoptosis in different cell lines, is constitutively cleaved in CGC, suggesting that its catalytic subunit is active per se. Moreover, KCl deprivation induces cyclin D1 expression and accumulation in nuclei. This process is blocked by bisindolylmaleimide. A model is proposed where, in the absence of survival signals, activated PKC delta induces cyclin D1 expression and accumulation in the nucleus, which subsequently, would lead to an aborted cell cycle and apoptosis.     

Hydrogen peroxide-induced motoneuron apoptosis is prevented by poly ADP ribosyl synthetase inhibitors

The impact of oxidative stress (H2O2) was observed using purified rat motoneuron cultures and H2O2-induced dose-dependent motoneuron death was demonstrated. The apoptotic characteristics of cell death were studied morphologically and using the TUNEL technique. This H2O2-induced motoneuron death was inhibited by the poly ADP ribosyl synthetase (PARS) inhibitors benzamide and nicotinamide. These findings suggest the potential utility of PARS inhibitors in the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis, in which oxidative stress has been suspected to play an important etiopathogenic role.     

Ca2+ changes and 86Rb efflux activated by hyposmolarity in cerebellar granule neurons

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl(-)-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl- and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]0, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels.     

Lithium protects rat cerebellar granule cells against apoptosis induced by anticonvulsants, phenytoin and carbamazepine

We have studied the neuroprotective actions of lithium against various insults in cultured cerebellar granule cells of rats. The anticonvulsants, phenytoin and carbamazepine, have been shown to induce apoptosis of cerebellar granule cells at high concentrations. Here we found that co-presence of LiCl (1-10 mM) dose-dependently protected against phenytoin (20 microM)- and carbamazepine (100 microM)-induced neuronal apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism, morphological inspection, chromatin condensation and DNA fragmentation. These neuroprotective effects were not prevented by inclusion of myoinositol nor mimicked by a potent inositol monophosphatase inhibitor, suggestive of a mechanism independent of inositol monophosphatase blockade. Lithium also significantly protected against apoptosis of cerebellar granule cells induced by aging of the cultures. Additionally, lithium suppressed death of cerebellar granule cells exposed to a low concentration of extracellular potassium. In contrast, it had no protective effect on cell death induced by Ca++ ionophores, a Na+ channel opener, a protein kinase inhibitor, a nitric oxide donor or H2O2. Thus, lithium has robust neuroprotective effects against apoptotic cell death induced by multiple insults with limited selectivity. These actions provide a new avenue to study the molecular and cellular mechanisms of this drug.     

Cytosine arabinoside induces apoptosis in cerebellar neurons in culture

Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC50 of approximately 60 microM when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC50 = 1 microM), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.